Oxidative stress induced by hydrogen peroxide disrupts zebrafish visual development by altering apoptosis, antioxidant and estrogen related genes

Hydrogen peroxide is considered deleterious molecule that cause cellular damage integrity and function. Its key redox signaling molecule in oxidative stress and exerts toxicity on a wide range of organisms. Thus, to understand whether oxidative stress alters visual development, zebrafish embryos were exposed to H2O2 at concentration of 0.02 to 62.5 mM for 7 days. Eye to body length ratio (EBR) and apoptosis in retina at 48 hpf, and optomotor response (OMR) at 7 dpf were all measured. To investigate whether hydrogen peroxide-induced effects were mediated by oxidative stress, embryos were co-incubated with the antioxidant, glutathione (GSH) at 50 μM. Results revealed that concentrations of H2O2 at or above 0.1 mM induced developmental toxicity, leading to increased mortality and hatching delay. Furthermore, exposure to 0.1 mM H2O2 decreased EBR at 48 hpf and impaired OMR visual behavior at 7 dpf. Additionally, exposure increased the area of apoptotic cells in the retina at 48 hpf. The addition of GSH reversed the effects of H2O2, suggesting the involvement of oxidative stress. H2O2 decreased the expression of eye development-related genes, pax6α and pax6β. The expression of apoptosis-related genes, tp53, casp3 and bax, significantly increased, while bcl2α expression decreased. Antioxidant-related genes sod1, cat and gpx1a showed decreased expression. Expression levels of estrogen receptors (ERs) (esr1, esr2α, and esr2β) and ovarian and brain aromatase genes (cyp19a1a and cyp19a1b, respectively) were also significantly reduced. Interestingly, co-incubation of GSH effectivity reversed the impact of H2O2 on most parameters. Overall, these results demonstrate that H2O2 induces adverse effects on visual development via oxidative stress, which leads to alter apoptosis, diminished antioxidant defenses and reduced estrogen production.


Maintenance of zebrafish
Zebrafish wildtypes (Danio rerio) were obtained from Academia Sinica and housed in 40-L tanks at a constant temperature of 28 °C, following a light cycle of 14 h of light and 10 h of darkness.They were fed a standard diet of commercial pellets daily and supplemented with brine shrimp twice a day at 9:00 am and 5:00 pm.To facilitate mating, adult wild-type females and males were placed in a mating box at a ratio of 2:1 and separated by a partition to acclimate to their environment.The following day at 9:00 am, the partition was removed, allowing the zebrafish to mate under light stimulation.Eggs were harvested 30 min post-spawning, subjected to washing www.nature.com/scientificreports/ in circulating system water to remove dead embryos and impurities.Fertilized eggs were then rinsed in embryo medium (EM) containing 0.004% CaCl2, 0.163% MgSO4, 0.1% NaCl, and 0.003% KCl.Subsequently, they were evenly distributed into 6-well plates, with 30 embryos placed in each well containing 8 mL of EM, and maintained at a constant temperature of 28 °C.All procedures involving zebrafish were conducted in accordance with local animal welfare regulations and approved by the Institutional Animal Care and Use Committee (IACUC) of NPUST (approval No. NPUST-105-067). 2 and glutathione (GSH) were obtained from Sigma Aldrich and prepared as stock solutions at concentrations of 100 mM and 10 mM, respectively, in distilled water.These stock solutions were then diluted with embryo medium (EM) to achieve the desired concentrations of H 2 O 2 (0.02, 0.1, 0.5, 2.5, 12.5, and 62.5 mM) for the experiment.Zebrafish embryos were exposed from 2 h post-fertilization (hpf) to 7 days post-fertilization (dpf) in 6-well plates, with 30 embryos in each well and three replicates per group.The exposed embryos were maintained at 28 °C, with exposure solutions changed daily.Dead embryos were removed immediately and zebrafish survival and hatching rates were observed every 24 h.

Eye and body length measurement
Zebrafish embryos at 48 hpf were used for eye to body length measurement.At 48 hpf, the zebrafish eye undergoes crucial development, forming key structures such as the lens, retina, and optic nerve.This stage is vital for early eye development and the detection of abnormalities 45 .According to published protocol 46 , the entire eye diameter was assessed as the distance between the pigmented epithelium of one pole to the opposite pole, aligned with the spine.Body length was measured from the snout tip to the end of the spine before the caudal.The eye-to-body length ratio was assessed under a microscope (Leica 58APO) from the lateral view, and the data were converted into millimeters (mm).Each group was examined with ten embryos, and the experiments were replicated three times using eggs obtained from distinct spawns.

Apoptosis cell assay
Apoptosis in the retina was conducted by using acridine orange (AO) staining at 48 hpf.Embryos were immersed in 5 µg/ml acridine orange (acridinium chloride hemi-[zinc chloride], Sigma Aldrich) in EM for 30 min and kept in the dark at 28 °C.Subsequently, embryos were rinsed repeatedly with EM, anesthetized with 0.016% tricaine, and positioned laterally before being mounted on a slide glass with 0.5% gel agarose.Apoptosis was examined using a fluorescent microscope (Leica M165 FC), with a focus on the eyes.The areas of apoptosis cell with fluorescent AO positivity were quantified using ImageJ software.Each group was examined with ten embryos, and the experiments were replicated three times using eggs obtained from distinct spawns.

Visual behavior assay
The optomotor response (OMR) was conducted for the visual behavior, which involves the movement of the head or body.OMR is effective in detecting abnormalities in visual function 47 .At 7 dpf, zebrafish larvae have developed enough to perform coordinated swimming movements, which makes it a suitable time point to assess motor activity and behavioral responses 48 .This method was adapted from a previously published protocol 49 .Zebrafish at 7 dpf were positioned in a specially designed petri dish containing five tracks (0.5 cm × 7 cm per track; Fig. 1) and were then incubated on a white screen for 30 s with light intensity at 825 nm.A study revealed that the visual behavior observed in zebrafish assays was responsive to light wavelengths spanning from 825 to 910 nm 50 .During the test, a white and black bars animation was presented moving both upward and downward at the same set of fixed speeds as a stimulus for the zebrafish, and the response was recorded for 30 s before and after measurement.All individuals moving in the following direction of the stimulus during the measurement are considered to demonstrate a positive OMR.The response of the zebrafish following the animation as indicated as positive OMR then measured in terms of swimming distance.Each group was examined with ten embryos, and the experiments were replicated three times using eggs obtained from different spawns.

Real-time PCR
Zebrafish embryos were exposed to nitrate and nitrite, and total RNA was extracted at 48 hpf (n = 20 per treatment group).Quantitative PCR was utilized to assess the expression levels of pair box protein (pax6α and pax6β), tumor protein 53 (tp53), caspase 3 (casp3), BCL2 associated X, apoptosis regulator a (bax), b-cell leukemia/ lymphoma 2α (bcl2α), superoxide dismutase-1 (sod1), catalase (cat), glutathione peroxidase 1a (gpx1a), estrogen receptor (ERs) (esr1, esr2α, and esr2β), ovarian and brain aromatases (cyp19a1a and cyp19a1b, respectively), and elongation factor 1 alpha 1 (eef1a1) were determined using quantitative PCR.The eef1a1 was used as an internal control.The specific primer used in this experiment are detailed in Table 1.Real-time PCR was carried out using KAPA SYBR FAST PCR reagent and an Applied Biosystems StepOnePlus Real-Time PCR system.The cycling profile included enzyme activation at 95 °C for 3 min, denaturation at 95 °C for 3 s, followed by annealing/primer extension for 40 cycles with denaturation at 95 °C for 3 s.

Ethics approval
The protocols for fish experiments were implemented according to local animal welfare regulations and approved (approval No. NPUST-105-067) by the Institutional Animal Care and Use Committee (IACUC) of NPUST.The study is reported in accordance with ARRIVE guidelines (https:// arriv eguid elines.org).

Statistical analysis
Statistical analysis of all data was conducted through one-way ANOVA, followed by Tukey's post hoc test, utilizing the SigmaPlot 12.5 package for Windows.A significance level of P < 0.05 was applied in all analyses to identify significant differences between the treatments.

Effects of H2O2 on survival and hatching rate
The study investigated the impact of different concentrations of H 2 O 2 on the survival of zebrafish embryos and larvae over time, as depicted in Fig. 2A.While 0.02 mM showed no toxicity, 0.1 mM and 0.5 mM H 2 O 2 showed toxicity, resulting in mortality rates of 14.4% and 37.8%, respectively, at 168 hpf.Higher concentrations of H 2 O 2 (2.5 mM, 12.5 mM, and 62.5 mM) resulted in 100% mortality, with survival declining rapidly from 24 to 168 hpf.Consequently, 0.02 mM, 0.1 mM, and 0.5 mM of H 2 O 2 will be further examined for their impact on zebrafish visual development.These results indicate that zebrafish development is sensitive to H 2 O 2 in a time-and dosedependent manner.
The hatching rates of zebrafish embryos exposed to varying concentrations of H 2 O 2 at different developmental stages are presented in Fig. 2B.The results indicated a dose-dependent effect of H 2 O 2 on the hatching rate under laboratory conditions.Compared to the control group, 0.02 mM of H 2 O 2 did not impact the hatching rate up to 168 hpf.However, 0.1 mM and 0.5 mM H 2 O 2 significantly delayed embryo hatching and exhibited toxicity.

Effects of H 2 O 2 on the eye to body length ratio
To determine whether H 2 O 2 has a deleterious effect on zebrafish visual development, embryos were exposed to varying concentrations of H 2 O 2 (0.02, 0.1, and 0.5 mM).As shown in Fig. 3A, exposure to 0.1 and 0.5 mM of H 2 O 2 significantly decreased the eye-to-body length ratio at 48 hpf, Interestingly, the reduction induced by 0.1 mM of H 2 O 2 was fully reversed upon the addition of GSH (Fig. 3B).

Effects of H 2 O 2 on the apoptosis in retina
An increase in fluorescence in the GFP channel correlates with increased AO staining and increased cell death in the embryo.Exposure to H 2 O 2 at concentrations of 0.1 and 0.5 mM resulted in an increased the fluorescent AO positive cells (Fig. 3C) and area of apoptotic cells in the retina at 48 hpf (Fig. 3E).Notably, the augmentation induced by 0.1 mM of H 2 O 2 was completely reversed upon the addition of GSH, as depicted in Fig. 3D,F.

Effects of H 2 O 2 on visual behavior
Exposure to H 2 O 2 affected zebrafish visual development at 48 hpf.To evaluate whether this change influenced visual behavior, OMR testing was conducted at 7 dpf.The results demonstrated a significant reduction in OMR swimming distance following exposure to 0.1 and 0.5 mM of H 2 O 2 , as depicted in Fig. 4A.The decrease induced by 0.1 mM of H 2 O 2 was completely reversed by the addition of GSH (Fig. 4B).

Effects of H 2 O 2 on gene expression
Exposure to H 2 O 2 at 0.1 mM significantly decreased the relative expression of genes associated with visual development, pax6α and pax6β at 48 hpf, and these effects were significantly reversed by addition of GSH (Fig. 5A,B).The relative gene expression of tp53, casp3 and bax significantly increased, while bcl2a was decreased, and these effects were also significantly reversed by the addition of GSH (Fig. 5C-F).As for antioxidant-related genes, exposure led to a significant decrease in the relative expression of sod1, cat and gpx1α, and these effects were significantly reversed by the addition of GSH (Fig. 5G-I).In terms of ERs, exposure to H 2 O 2 significantly reduced the relative expression levels of esr1, esr2α, and esr2β, with the effects reversed upon GSH addition (Fig. 5J-L).Similarly, exposure resulted in a significant reduction in the relative expression levels of aromatase genes, cyp19a1a and cyp19a1b, which were also reversed by the addition of GSH (Fig. 5M,N).

Discussion
Our study demonstrated the adverse impacts of H 2 O 2 exposure on various aspects of zebrafish visual development.
We observed detrimental effects on survival and hatching rate, eye to body length ratio, apoptotic cell in the retina, OMR swimming behaviour and genes related to eye development, apoptosis, antioxidant and estrogen signalling.Notably, our findings revealed a dose-dependent relationship, with significant effects observed starting from a concentration of 0.1 mM H 2 O 2 .Another study in common carp showed tissue-specific responses to H 2 O 2 exposure, affecting immune, inflammatory, autophagic, and DNA damage pathways at 0.2 mM to 1 mM H 2 O 2 concentrations 51 .Additionally, exposure to H 2 O 2 concentrations ranging from 0 to 100,000 nM impacted the locomotion and metabolism of both zebrafish larvae and adults 52 .It's noteworthy that this concentration range falls within the spectrum of H 2 O 2 levels found in aquatic environments such as lake, river, rain, estuary, and open ocean, ranging from 0.004 μM to 199 μM 9 .During the algal bloom period, levels exceeding 10,000 nM of H 2 O 2 have been observed 53 .These results indicate the potential ecological relevance and implications of H 2 O 2 -induced toxicity in aquatic organisms like zebrafish.
To ascertain whether an eye is proportionally large relative to the subject's overall body size, eye to body length ratio were measured.Exposure to H 2 O 2 resulted in a reduction of the eye-to-body length ratio in zebrafish at 48 hpf.This aligns with our observations, as previous research has utilized zebrafish to assess body length ratios, indicating that eye size is indeed proportionally correlation to overall body size 46 .Similarly, in other species, eye size has been shown to be tightly regulated relative to overall body size 54,55 , yet many animal models with eye defects exhibit abnormal body sizes 56 .Therefore, the decrease in eye size observed in zebrafish exposed to H 2 O 2 may be correlated with a reduction in body length.This correlation suggests that the effects of H 2 O 2 exposure extend beyond the visual system, potentially impacting overall growth and development in zebrafish.It's plausible that the oxidative stress induced by H 2 O 2 could disrupt normal physiological processes, leading to alterations in both eye size and body length.
Apart from its significance as a biological phenomenon, the detection of apoptotic processes has been implicated in a wide range of diseases.Our research findings indicate that exposure to H 2 O 2 resulted in an increase in the positive area of apoptotic cells in the retina of zebrafish at 48 hpf, as observed through AO staining.AO is a fluorescent dye binds to nucleic acids, specifically DNA and RNA, by intercalating between the base pairs and is utilized to investigate apoptosis in zebrafish and other model organisms.This property allows AO to stain DNA and RNA molecules, making them visible under a fluorescence microscope 57 .The observed increase in the positive area of AO fluorescence cells following exposure to H 2 O 2 is correlated with elevated cell death.Thus, our study demonstrates that exposure to H 2 O 2 leads to increased apoptotis in zebrafish retina at 48 hpf.
To investigate the impact of H 2 O 2 exposure on visual behavior, we conducted the OMR assay.The OMR is an innate visuomotor reflex observed in zebrafish, where the fish aligns its swimming direction with a highcontrast visual stimulus to stabilize its position relative to the stimulus.This assay is commonly employed to assess changes in vision-related behaviors 58 .Utilizing the OMR task in zebrafish has proven effective in detecting visual abnormalities induced by genetic mutations and toxin exposure 59,60   Exposure significantly altered relative genes expression involved in eye development, apoptosis, antioxidant and estrogen signalling were assessed by qPCR at 48 hpf.Pax6 is essential for the development of the eye in numerous species, including flies, zebrafish, mice, and humans 61 .Furthermore, pax6 is expressed in neuronal progenitor cells within the regeneration process of the adult zebrafish retina 62,63 .Zebrafish possess two paralogous pax6 genes, pax6α and pax6β, which encode functionally redundant proteins responsible for regulating the formation and differentiation of the retina and lens 64 .The knockdown of either pax6α and pax6β resulted in the disruption of retinal regeneration in zebrafish 65 .Reduced expression of pax6 has been associated with the loss of eye function 66 .Our results align with this finding, demonstrating a significant decrease in the expression of both pax6α and pax6β following exposure to H 2 O 2 .This suggests that the exposure may impair eye function, potentially contributing to the abnormalities observed in visual behavior as detected by the OMR assay.
Our study demonstrated that exposure to H 2 O 2 elevated cell death in the retina at 48 hpf.The tp53 gene is a well-known tumor suppressor gene responsible for regulating both cell proliferation and cell death in response to www.nature.com/scientificreports/DNA damage 67 .Various DNA-damaging agents have been demonstrated to elevate zebrafish tp53 transcription 68 and enhance tp53 protein levels 69 .There is substantial evidence indicating that death signals triggered by tp53 lead to the activation of caspases.Multiple studies have been carried out to clarify the function of caspase activation in cell death mediated by tp53 [70][71][72][73] .Transcripts for caspase-3 are present during early embryogenesis 74 .Additionally, caspase-3 is a key player in the advancement of apoptosis, resulting in changes in cell morphology and DNA fragmentation.Our study demonstrated that exposure to H 2 O 2 increased tp53 and caps3, indicating that cell death induced by H 2 O 2 follows an apoptotic mechanism.This implies that the cellular response to H 2 O 2 involves the activation of apoptotic pathways mediated by tp53 and caps3, which play crucial roles in regulating programmed cell death.Moreover, mitochondria play a significant role in one of the primary pathways for caspase activation.Bcl-2 suppresses apoptosis and enhances cell survival, while bax operates within the mitochondria to trigger the production of cytochrome c, initiating the caspases activation 75 .In the present study, H 2 O 2 reduced bcl2α expression and increased bax, indicating the involvement of mitochondria in H 2 O 2 -induced apoptosis, leading to developmental defects in the visual system of zebrafish.H 2 O 2 has the capacity to initiate apoptosis via diverse mechanisms through generating oxidative stress 76 .Oxidative stress arises from an imbalance between ROS production and the ability of the body's antioxidant defenses to neutralize them.In this study, H 2 O 2 exposure induced alterations in antioxidant-related genes such as sod1, cat, and gpx1a.SOD1, part of the SOD family, crucial endogenous antioxidant enzymes that serve as the primary defense against ROS within cells.It catalyses the dismutation of superoxide into H 2 O 2 and oxygen, thus reducing the harmful effects of superoxide anion.Catalase or glutathione peroxidase detoxify H 2 O 2 and convert it into water.Catalase efficiently breaks down H 2 O 2 , while glutathione peroxidase also aids in converting H 2 O 2 into water and oxygen.Glutathione peroxidases, particularly GPx1, have been associated with both the onset and prevention of various common and complex diseases 77 .Studies have shown that gpx1 provide protection against apoptosis triggered by oxidative stress 78 , while also decreasing the expression bax (pro-apoptotic protein) 79 .We demonstrated that H 2 O 2 reduced the expression of sod1, cat, and gpx1a, while increased the pro-apoptotic gene.This suggests a decrease in antioxidant capacity and an increased vulnerability to oxidative stress, which could trigger apoptosis, or programmed cell death, potentially contributing to visual dysfunction during zebrafish development.
Oxidative stress plays a significant role in influencing E2 signaling pathways by modulating the activity of estrogen receptors (ERs) 80 .E2, crucial for various physiological functions, is synthesized from testosterone through the action of the aromatase enzyme 81 .In zebrafish, two distinct aromatase genes are present: cyp19a1a, which encodes ovarian aromatase found in the gonads, and cyp19a1b, which encodes brain aromatase found in neural tissues such as the brain and retina 36,82 .The physiological effects of estrogen primarily occur through its binding to two types of ERs.Zebrafish possess two types of ERs, namely Erα and Erβ, which are encoded by three distinct genes: esr1, esr2α, and esr2β 83 .While E2 might possess an essential role in maintaining eye health, the exact mechanisms are still not fully understood.Nevertheless, hormone replacement therapies have been linked to lower rates of eye diseases like glaucoma, AMD, and cataracts, indicating that estrogen could be important for eye health 84 .The impact of oxidative stress induced by H 2 O 2 exposure on the expression of ERs (esr1, esr2α, and esr2β) and aromatases (cyp19a1a and cyp19a1b) in zebrafish was examined.Our results demonstrated that H 2 O 2 decreased the expression of ERs (esr1, esr2α, and esr2β) and aromatases (cyp19a1a and cyp19a1b), likely due to the influence of excessive oxidative stress production induced by H 2 O 2 .Previous studies have highlighted the pivotal role of E2 in eye development and function, as evidenced by the presence of aromatase in the retina and ERs distributed across various retinal layers in different vertebrate species 85,86 .The oxidative stress induced by H 2 O 2 exposure may lead to decreased estrogen production, potentially impacting visual function in zebrafish, given the importance of estrogen for maintaining visual development.Therefore, understanding the impact of oxidative stress on estrogen-related pathways is critical for elucidating the mechanisms underlying visual development and potential implications for visual disorders.
Interestingly, co-incubation of GSH effectivity reversed the impact of H 2 O 2 on most parameters, suggesting that oxidative stress mediates these effects.Glutathione serves as a vital antioxidant pivotal in mitigating oxidative stress within cellular environments.Its principal function involves the neutralization of ROS and shielding cells from oxidative damage 77 .This principal function of GSH involves the scavenging of ROS, including hydroxyl radicals ( • OH), superoxide radicals (O 2 •-), and peroxyl radicals (ROO • ), which are generated during oxidative stress conditions.By donating an electron to these highly reactive species, GSH effectively neutralizes them and prevents them from causing oxidative damage to cellular structures such as lipids, proteins, and DNA 87 .Prior research indicates that providing glutathione helps protect cells from oxidative stress-induced damage 88 .Conversely, reduced levels of glutathione, which lead to the impairment of the glutathione-dependent enzyme pathway, are linked to the development and advancement of numerous diseases 89 .Therefore, the addition of GSH unequivocally illustrated that the impact of H 2 O 2 on zebrafish visual development is mediated through oxidative stress.In some cases, GSH alone alter genes expression, suggesting that antioxidants might affect gene regulation by modulating pathways associated with the response to oxidative stress.Numerous investigations have also demonstrated that bursts of oxidant generation, as well as significant variations in the responses of several antioxidant defences, are strongly related with changes in gene expression across a range of tissues from genetically distinct organisms 90,91 .GSH effectively mitigates oxidative stress and reduces the risk of cellular damage and apoptosis 77 .Therefore, the protective effects of GSH in zebrafish exposed to H 2 O 2 are attributed to its dual role in free radical quenching and antioxidant activity, which collectively help maintain cellular integrity and promote survival under oxidative stress conditions.

Conclusion
In summary, our study showed that exposure to H 2 O 2 triggers oxidative stress, resulting in adverse effects on the visual development of zebrafish, including changes in eye-to-body length ratios, apoptotic activity in the retina, and altered OMR responses.These alterations are linked to the excessive oxidative stress, resulting in apoptosis, antioxidant imbalance, reduced estrogen production, and impaired zebrafish visual development.These findings demonstrate the critical role of oxidative stress in mediating adverse effects on visual function and development, emphasising the importance of antioxidant defenses in protecting against such harmful effects.

Figure 1 .
Figure 1.Apparatus for visual behavior assessment of OMR.Arrows (→) refers to the direction of animation.The experiments were conducted in the dark place at room temperature.

Figure 2 .
Figure 2. Survival (A) and hatching rate (B) of zebrafish embryos were measured following exposure to various concentration of H 2 O 2 for 168 hpf.Each value is expressed as the mean ± SD of three replicates.

Figure 3 .
Figure 3. Effects of exposure to H 2 O 2 and co-exposure to GSH on visual system.Eye to body length ratio was measured at 48 hpf: exposure to 0.02, 0.1, and 0.5 mM H 2 O 2 (A), and co-exposure to 50 μM GSH with 0.1 mM H 2 O 2 (B) (n = 10).Representative image of lateral views of apoptotic cells within the retina of live zebrafish embryos was observed at 48 hpf using AO staining: exposure to 0.02, 0.1, and 0.5 mM H 2 O 2 (C), and co-exposure to 50 μM GSH with 0.1 mM H 2 O 2 (D).Apoptosis signals were indicated by green fluorescent spotted (dot) on the retina.Area of apoptotic cells in the retina was measured at 48 hpf: exposure to 0.02, 0.1, and 0.5 mM H 2 O 2 (E), and co-exposure to 50 μM GSH with 0.1 mM H 2 O 2 (F) (n = 10).Each value is expressed as the mean ± SD.Different symbols in each graph indicate significant differences (p < 0.05).The experiments were repeated 3 times with different cohorts of eggs.

Figure 4 .Figure 5 .
Figure 4. Effects of exposure to H 2 O 2 and co-exposure to GSH on OMR assay.OMR was measured at 7 dpf: exposure to 0.02, 0.1, and 0.5 mM H 2 O 2 (A), and co-exposure to 50 μM GSH with 0.1 mM H 2 O 2 (B) (n = 10).Each value is expressed as the mean ± SD.Different symbols in each graph indicate significant differences (p < 0.05).The experiments were repeated 3 times with different cohorts of eggs. https://doi.org/10.1038/s41598-024-64933-5www.nature.com/scientificreports/ . Our study revealed that exposure to H 2 O 2 resulted in reduced swimming behavior in response to the positive directions of the OMR stimulus.This finding implies that defects caused by H 2 O 2 exposure may contribute to abnormal visual behavior, suggesting a potential association between H 2 O 2 exposure and visual impairments.